We use HEK cells for lentiviral transfection, our vector has Gfp marker so as a routine we always check our cells under fluorescent microscope 4 days after transfection. The last time our HEK cells didn't seem healthy and happy after thawing them from liquid nitrogen Tank and -80 freezer. But they finally attached well and seemed healthy after 3 passages and they were fine prior to transfection process. After 4 days we look them up under fluorescent microscope and we saw the attached pictures, it seems our cells have distinct boundaries and our gfp percentage went much lower than we expected. As you can see in attached pictures a population of our cells are gfp+ and other population are gfp negative. Does anyone have any similar problems with HEK cells?
In addition, what is the appropriate passage number for transfection after thawing HEK cells? Is there a maximum or minimum passage number?
Can you take pictures in higher magnification and add them here? The cells do not seem healthier, probably dried.
I agree with Chandra, these cells do not look healthy. If possible, I would thaw a new batch of cells and start over. In my experience, HEK cells are fairly robust and transfect well. They often plate well after thawing, and I routinely transfect after only passaging them once. They do not seem to like low-serum media, so you could check the makeup of your media. We use 10% FBS they grow well in that.
Contamination? Cell line crosscontamiation? need bigger magnification
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